Cucumber mosaic virus coat protein modulates the accumulation of 2b protein and antiviral silencing that causes symptom recovery <i>in planta</i>

<div><p>Shoot apical meristems (SAM) are resistant to most plant viruses due to RNA silencing, which is restrained by viral suppressors of RNA silencing (VSRs) to facilitate transient viral invasion of the SAM. In many cases chronic symptoms and long-term virus recovery occur, but the underlying mechanisms are poorly understood. Here, we found that wild-type <i>Cucumber mosaic virus</i> (CMV^WT) invaded the SAM transiently, but was subsequently eliminated from the meristems. Unexpectedly, a CMV mutant, designated CMV^RA that harbors an alanine substitution in the N-terminal arginine-rich region of the coat protein (CP) persistently invaded the SAM and resulted in visible reductions in apical dominance. Notably, the CMV^WT virus elicited more potent antiviral silencing than CMV^RA in newly emerging leaves of infected plants. However, both viruses caused severe symptoms with minimal antiviral silencing effects in the <i>Arabidopsis</i> mutants lacking host RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) or SUPPRESSOR OF GENE SILENCING 3 (SGS3), indicating that CMV^WT induced host RDR6/SGS3-dependent antiviral silencing. We also showed that reduced accumulation of the 2b protein is elicited in the CMV^WT infection and consequently rescues potent antiviral RNA silencing. Indeed, co-infiltration assays showed that the suppression of posttranscriptional gene silencing mediated by 2b is more severely compromised by co-expression of CP^WT than by CP^RA. We further demonstrated that CP^WT had high RNA binding activity leading to translation inhibition in wheat germ systems, and CP^WT was associated with SGS3 into punctate granules <i>in vivo</i>. Thus, we propose that the RNAs bound and protected by CP^WT possibly serve as templates of RDR6/SGS3 complexes for siRNA amplification. Together, these findings suggest that the CMV CP acts as a central hub that modulates antiviral silencing and VSRs activity, and mediates viral self-attenuation and long-term symptom recovery.</p></div

A model explaining the CMV CP-modulated conflict between RNA silencing and 2b-induced suppression in the shoot apices infected with CMV^WT.

<p>During the early infection stages of CMV^WT, levels of CP^WT accumulation are not sufficient to affect accumulation of 2b protein and antiviral silencing. However, at intermediate stages of replication, higher CP^WT levels accumulate and bind to viral RNAs to result in virion assembly, reduced 2b protein accumulation and protection of aberrant RNAs from degradation. The CP^WT and bounded RNA complex are postulated to recruit RDR/SGS3 for amplification of vsiRNAs that increase antiviral RNA silencing and antagonize 2b protein suppression of host RNA silencing. In summary, CP^WT RNA binding inhibits the translation and accumulation of the 2b protein to favor RNA silencing that contributes to viral self-attenuation and long-term symptom recovery.</p

Image_4_Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus.JPEG

<p>The most economically important plant viruses are specifically transmitted by phytophagous insects that significantly affect viral epidemiology. Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus, is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent-propagative manner. However, the infection route of BYSMV in SBPHs is poorly understood. In this study, immunofluorescence confocal laser scanning microscopy (iCLSM) was performed to investigate the route of BYSMV in SBPHs. We unexpectedly found that BYSMV initially infected the hindgut epithelium of SBPHs, instead of the midgut epithelium initially infected by other persistent-propagative viruses. Subsequently, BYSMV disseminated to the hindgut visceral muscles and spread to other parts of alimentary canals, hemolymph, and salivary glands. Comparative analysis of gene expression on viral mRNAs and the BYSMV nucleoprotein by using different molecular detection and immunohistochemistry further demonstrated that BYSMV initially infected and replicated in the hindgut epithelial cells of SBPHs. Collectively, our study provides the first insight into that hindgut is initial infection site of BYSMV that represents a new dissemination route of persistent-propagative viruses.</p

CMV CP attenuates 2b-mediated suppression of local GFP silencing.

<p>(A) GFP fluorescence in regions of <i>N</i>. <i>benthamiana</i> leaves after agroinfiltration of sGFP reporter gene (OD₆₀₀ = 0.4), in combination with different amounts of the pGD empty vector (V, OD₆₀₀ = 0.4), the pGD-2b (OD₆₀₀ = 0.2) and the pGD-CP vectors (OD₆₀₀ = 0.4), as indicated. Photographs were taken under UV light at 5 dpi. (B) Protein gel blot analysis of samples extracted from infiltrated region shown in panel A. (C) Accumulation of GFP mRNA and siRNAs in the region shown in panel A. Methylene blue-stained rRNA and U6 RNA were used as loading controls for high and low molecular weight RNAs, respectively. The values under GFP mRNA (RA1) and siRNAs (RA2) represent the relative accumulation (RA) of GFP mRNA and GFP-derived siRNAs, respectively. The RA values of sGFP with 2b and V were set as 1. RA2/RA1 ratios under U6 detection represent the relative production of GFP-derived siRNAs versus GFP mRNA. (D) GFP fluorescence (left panel) in local patches agroinfiltrated with the sGFP vector (OD₆₀₀ = 0.4), empty pGD vector, and the 2b vectors (OD₆₀₀ = 0.2), either alone or in combination with CP^WT or CP^RA vectors (OD₆₀₀ = 0–0.8), as indicated in middle panel. A protein gel blot analysis of samples extracted from the infiltrated regions is shown in the right panel. Anti-GFP, -CP, and -2b polyclonal antibodies were used to assess the GFP, CP, and 2b, accumulations, respectively. Mock-infected plants were used as the negative control. Coomassie brilliant blue (CBB) staining was used as the protein loading control.</p

Image_1_Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus.JPEG

<p>The most economically important plant viruses are specifically transmitted by phytophagous insects that significantly affect viral epidemiology. Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus, is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent-propagative manner. However, the infection route of BYSMV in SBPHs is poorly understood. In this study, immunofluorescence confocal laser scanning microscopy (iCLSM) was performed to investigate the route of BYSMV in SBPHs. We unexpectedly found that BYSMV initially infected the hindgut epithelium of SBPHs, instead of the midgut epithelium initially infected by other persistent-propagative viruses. Subsequently, BYSMV disseminated to the hindgut visceral muscles and spread to other parts of alimentary canals, hemolymph, and salivary glands. Comparative analysis of gene expression on viral mRNAs and the BYSMV nucleoprotein by using different molecular detection and immunohistochemistry further demonstrated that BYSMV initially infected and replicated in the hindgut epithelial cells of SBPHs. Collectively, our study provides the first insight into that hindgut is initial infection site of BYSMV that represents a new dissemination route of persistent-propagative viruses.</p

RNA binding and translation inhibition by the CP^WT and CP^RA proteins.

<p>(A) RNA-binding abilities of the GST-CP^WT and GST-CP^RA proteins as assessed by digoxigenin-labeled CMV RNA4 or Luciferase (Luc) mRNA (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006522#sec011" target="_blank">Materials and methods</a> for details). GST served as a negative control. Coomassie brilliant blue (CBB) staining was used as the protein loading control. (B) Translation inhibition of Luciferase mRNA by CP^WT and CP^RA <i>in vitro</i>. The Luc mRNA was transcribed and incubated with different concentrations of the GST, GST-CP^RA, or GST-CP^WT in wheat germ extract at 25°C for two hours. Then, the activity of translated Luciferase <i>in vitro</i> was measured with a luminometer. Luciferase activities from mRNA without GST, GST-CP^RA, or GST-CP^WT was set as 100%. Error bars represent the standard error of the mean. Data points are the mean value of three independent experiments. *<i>P</i>-value < 0.05; ** <i>P</i>-value < 0.01; *** <i>P</i>-value < 0.001.</p

CMV^WT induces more potent antiviral silencing than CMV^RA in emerging <i>N</i>. <i>benthamiana</i> leaves.

<p>(A) Accumulation of CMV gRNAs and sgRNAs and (B) RNA3-vsiRNAs in emerging <i>N</i>. <i>benthamiana</i> leaves at 7 dpi with CMV^RA and CMV^WT. Loading controls for the high and low molecular weight RNAs were rRNA and U6 RNAs, respectively. (C) Ratios of accumulation levels of CMV gRNA3 and RNA3-vsiRNAs calculated from signal intensities in three independent hybridization experiments. The vsiRNAs/RNA3 values refer to the relative ratios of RNA3-vsiRNAs versus viral genomic RNA3. The values of RNA3 and RNA3-vsiRNAs in CMV^RA-infected leaves were set as 1. ** <i>P</i>-value < 0.01; *** <i>P</i>-value < 0.001.</p

Image_3_Transmission Characteristics of Barley Yellow Striate Mosaic Virus in Its Planthopper Vector Laodelphax striatellus.JPEG

<p>The most economically important plant viruses are specifically transmitted by phytophagous insects that significantly affect viral epidemiology. Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus, is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent-propagative manner. However, the infection route of BYSMV in SBPHs is poorly understood. In this study, immunofluorescence confocal laser scanning microscopy (iCLSM) was performed to investigate the route of BYSMV in SBPHs. We unexpectedly found that BYSMV initially infected the hindgut epithelium of SBPHs, instead of the midgut epithelium initially infected by other persistent-propagative viruses. Subsequently, BYSMV disseminated to the hindgut visceral muscles and spread to other parts of alimentary canals, hemolymph, and salivary glands. Comparative analysis of gene expression on viral mRNAs and the BYSMV nucleoprotein by using different molecular detection and immunohistochemistry further demonstrated that BYSMV initially infected and replicated in the hindgut epithelial cells of SBPHs. Collectively, our study provides the first insight into that hindgut is initial infection site of BYSMV that represents a new dissemination route of persistent-propagative viruses.</p

Distribution of CMV^RA or CMV^WT in the apical meristems of infected plants.

<p><i>In situ</i> hybridization of longitudinal sections of shoot apices in <i>N</i>. <i>benthamiana</i> plants inoculated with CMV^RA and CMV^WT at 7-, 14-, and 21- dpi. Dark areas indicate the presence of viral RNA revealed by digoxigenin-labeled riboprobe corresponding to the CMV CP. CMV signals were not observed in the shoot meristem from uninfected samples (left panel). Only a low signal density was present in CMV^WT infected meristems at 7 dpi and these disappeared from the meristem by 14 dpi (middle panels). In contrast, CMV^RA infection contained abundant signal densities beneath the meristems at 7 dpi, and indicated partially invaded meristems by 14 dpi, and completely invaded meristems at 21 dpi (right panels). Bars = 100 μm.</p

CMV CP is associated with RDR6/SGS3 complex.

<p>(A) Interactions detection of SGS3 with CP^WT or CP^RA in <i>N</i>. <i>benthamiana</i> leaves epidermal cells using bimolecular fluorescence complementation (BiFC). Leaves were infiltrated with pairs of Agrobacterium strains harboring tested proteins tagged with the different halves of YFP. Rubisco protein (Rub) was negative control. Images were taken at 2 dpi by confocal scanning laser microscopy. Bars represent 20 μm. (B) Co-immunoprecipitation (Co-IP) assays showing associations of SGS3 with CPs <i>in vivo</i>. <i>N</i>. <i>benthamiana</i> leaves coexpressing Flag-CP^WT and Flag-CP^RA proteins in combination with the GFP-SGS3 protein were homogenized and incubated with anti-Flag M2 antibodies for co-IP assays. GFP was included as a negative control. Anti-GFP and anti-Flag antibodies were used to detect accumulations of GFP-SGS3/GFP and CP, respectively. The positions of GFP-SGS3 and GFP are indicated by black and white triangles, respectively.</p